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Effect of UV-B irradiation on release of arachidonic acid from B-16 melanoma cells
Prostaglandins Leukotrienes and Essential 0 Longman Group UK Ltd 1989
Fatty
Acids
(1989) 35. 153-156
Effect of UV-B Irradiation on Release of Arachidonic Acid from B-16 Melanoma cells T. SUEMATSU,
T. HIDAKA,
T. SAKANASHI,
Department of Medical Biochemistry, Kurume, 830, Japan (Reprint request:
M. SUGIYAMA
Kurume University to R. 0.)
School
and Ft. OGURA of Medicine,
67 Asahi-machi,
Abstract - B-16 melanoma cells in culture were prelabeled with (3H)-arachidonate, and exposed to UV radiation. Immediately after irradiation the cells released labeled materials. This UV-stimulated release was inhibited by mepacrine (20 PM) and calmodulin inhibitor W7 (0.5 PM). To determine the influence of extracellular Ca2+ on the UV-stimulated release, experiments were made with media containing various concentrations of Ca”. The release decreased significantly at lower Ca*+ concentrations. These results suggest that Ca*+-calmodulin-dependent phospholipase A2 was involved in UV-stimulated release
of radiolabeled
materials,
possibly
arachidonic
acid and its metabolites,
Introduction
Plasma membranes are among the cell constituents most sensitive to oxidative damage because of their high content of polyunsaturated fatty acids mainly esterified in the 2-position of various cellular phospholipids. In cultured B-16 melanoma cells, lipid peroxides promptly’ increase after UV exposure but 6 hours later the content of lipid peroxides drops to less than the control level (1). This phenomenon suggests that cultured B-16 melanoma cells have some mechanism which reduces lipid peroxides. Phospholipase A2 hydrolyzes ester bounds in the 2-position of membrane phospholipids and releases free fatty acids. There is much evidence that the GSH-peroxidase-GSH-reductase system can not directly act on fatty acyl chains which are esterified in membrane phospholipids (2), and
from the cells.
that phospholipase AZ is involved processes of membrane phospholipids by oxidative stress (3). In this study, B-16 melanoma cells were exposed to UV-B irradiation as tive stress and a release of arachidonic examined. Materials
in repair damaged in culture an oxidaacid was
and Methods
Cell culture
A line of B-16 melanoma cells was a gift from Shiseido Co. Ltd (Tokyo). B-16 melanoma cells were cultured at 37°C in a 5% COd95% air atmosphere in Eagle’s minimal essential medium (Eagle’s MEM, Nissui) supplemented with 10% calf serum (Flow Laboratories). Cells were harvested using 0.02% EDTA / C*+- Mg*+-free
153
1.54
PROSTAGLANDINS
Dulbecco’s phosphate buffered saline ( PBS(-)) and 0.25% tripsin (9/l, v/v). Cells (2 x 105/dish) were plated as monolayers in Q 60 mm dishes, usually 48 hrs prior to experiments. Following experiments were performed when the number of cells reached approximately 1 x 10h/dish. Cell viability was confirmed by the trypanblue exclusion test. Ultraviolet (UV) irradiation of the cells
Approximately 1 x lo6 cells in a monolayer were irradiated with four UV lights (Toshiba, FL-20, SE 30, wave length 290-320 nm UV-B). Release of arachidonic after W-B exposure
acid and its metabolites
After 24 hrs incubation following the cell seeding, the seeding media were removed and the cells were labeled with 0.4 /_Ki [‘Hl-arachidonic acid (“H)AA) (specific activity: 155 Ci/mol, Amersham International) in 4 ml of MEM + 10% calf serum. The labeling media were removed after 24 hr incubation in a 5% CO2/95% air atmosphere, and the cells were washed twice with 1 ml of incubaton media consisting of PBS(-) containing 0.1% glucose, 0.05% fatty acid-free bovine serum albumin and 1 mM CaC&. Then 1.2 ml of the incubation media was added again. After 1 hr incubation ior stabilization, a 0.2 ml portion of the incubation media was removed to measure each initial [‘HI background. Thereafter the dishes were irradiated for 15 min (1600 J/m’). Some of the dishes were protected from the irradiation by being covered with aluminium foil as a control. Immediately after UV exposure, the incubation media were collected from the dishes. The media obtained before and after irradiation were centrifuged at 3000 rpm for 10 min to remove detached cells and mixed with 10 ml of liquid scintillation liquid (toluene containing 35% (V/V) Triton X-100, 5 $I 2,5diphenyloxazole and 0.43 $1 1,4-bis-2,5-phenyloxazole benzene), and then assayed by a liquid scintillation counter (Aloka, LSC-673). Radio activities of [3H]AA and its metabolites released during the 15 min exposure were determined by calculating the difference of radioactivities before and after the exposure. Effects of the following treatments were evaluated by the ratio of the radioactivity from UV-exposed cells to that from UVprotected cells.
LEUKOTRIENES
AND ESSENTIAL FA-I-I’Y ACIDS
Study in few Ca’+ condition
In order to study the effect of extracellular Ca?+ concentration on the release of arachidonic acid and its metabolites stimulated by UV-B, four different media, which contained 1 mM Ca’+, 20 I_LMCa’+, 5 PM (a’+, or 5 PM Ca’+ + 6 PM EGTA respectively, were used. The concentration of Ca’+ was confirmed with a Hitachi atomic absorption spectrophotometer (180-60/80). Inhibitor studies
Effects of phospholipase inhibitor and calmodulin inhibitors on UV-B-induced release of arachidonic acid and its metabolites were examined by the addition of 20 PM mepacrine, 0.5 PM W7 or W12 to the medium, These inhibitors did not affect cell viability after 15 min UV exposure. Results B-16 melanoma cells prelabeled with (“H)AA were exposed to various doses of UV. Radiolabeled material(s) was released into the extracellular medium in a UV-dose dependent fashion. However, cell viability markedly decreased with increasing UV-B dose. The maximum energ which did not affect viability was 16 000 J/m Y, Average release at 16 000 J/m* of UV was significantly higher than that of unexposed controls (p 95%) was extracted with chloroform: methanol (2: 1, v/v), and 91% of the extracted radioactivity was due to free (‘H)AA, as determined by thin-layer chromatography. Table 1 Total Radioactivity of (‘H) Arachidonic Acid and Related Materials Released from B-16Melanoma Cells Following 1.5 min UV-B Exposure UV exposure Control
*= p > 0.05.
1366 * 698 742 f 542
Values are mean + SD.
dpm
*
dpm (n = 13)
Table 2 (I, II, III and IV) shows the influence of lowering the concentration of extracellular Ca’+ on the release of the radiolabeled materials. The UV-stimulated release significantly decreased with decreasing concentration of Cal+. Table 2 (I, V, VI and VII) shows the inhibitory effects of mepacrine (a phospholipase inhibitor) and W7 (a calmodulin inhibitor) on the release
EFFECT OF UV-B IRRADIATION
ON RELEASE
OF ARACHIDONIC
Effects of Extracellular Ca’+ Concentrations, the Calmodulin Inhibitor W7, and Mepacrine on the Total Release of (3H) Arachidonic Acid and Related Materials from B-16 Melanoma Cells
Table 2
Treatment Control fI mM Ca”) 20 /LM (‘a” 5 /.LM Ca” 5 /LM Ca’+ + 6 ELM EGTA Mepacrine20 PM (1 mM Ca”) WI2 0.5 PM (1 mM Ca”) W7 0.5 +M (1 mM Ca”)
UV-induced / protected from UV 2.30 + 1.25 (n = 13) 1.72 1.17 0.94 1.20
rt 2 k k
0.65 0.59 0.59 0.22
(n (n (n (n
= = = =
12) 9)* 6)* 5 6)*
2.43 f 0.81 (n = 7) 1.26 f (1.44 (n = 7)* 1
(UV-treated/protected from UV) indicates the ratio of the radioactivity from UV-exposed cells to that from UVprotected cells. n = number of experiments. * = p < 0.05 as compared with the control. 6 = p < 0.05 as compared with the group treated with 20 /LM Cal+. 1 = p < 0.01 as compared with the group treated with W17.
of the radioactive materials. In the presence of the release was inhibited 20 PM mepacrine, significantly (p
Our confirmations in this study include: (I) UVB irradiation stimulated the release of radiolabeled materials from (3H)AA-prelabeled B-16 melanoma cells; (II) the release of radiolabeled materials was dependent on extracellular Ca’+ concentrations; (III) the release was inhibited by the phospholipase inhibitor, mepacrine, and (IV) the release was inhibited by the specific calmodulin inhibitor, W-7. Phosphoglycerides were the main lipids incorporating radiolabeled AA. The released materials must include not only AA but also eicosanoids and other oxidized lipids derived from AA. The release is most likely due to the activation of phospholipase AZ, which is thought to be an enzyme universally occuring in live cells. Our preliminary experiment also confirmed the activity of this enzyme in B-16 melanoma
ACID FROM B-16 MELANOMA
CELLS
155
homogenate (19.2 5 12.0 nmol oleic acid release / mg protein / hr). Phospholipase Al has been established to be dependent upon intracellular Ca2+ concentrations except in lysosomes in which it is rather inhibited by the ion [4,5]. Our result that the UV-stimulated release was more inhibited at lower extracellular Ca” concentrations suggests that the UV-B irradiation perturbed the plasma membrane in favor of increasing the influx of Ca’+, and resultantly activated the involved enzyme, probably phospholipase A,, in an intracellular-Ca’+-conccntration dependent manner. This view agrees with Deutike. et al, who reported that oxidativc damage by t-butylhydroperoxide formed an ionpermiable leak pathway, probably due to phospholipid peroxidation (6). since UV-B is an oxidative stress already shown to trigger lipid peroxidation in cultured B-16 melanoma cells (1). The fact that mepacrine could markedly inhibit the UV-B activated release strongly suggests that phospholipase A: was involved in the release of radiolabelled materials by UV-B irradiation. As concerns the calmodulin inhibitor. WCwere obliged to use the rather low concentration of W7, because UV-B exposure to cells in the presence of more than 0.5 PM W7 lead to drastically low viability (84% or 36% viability in 0.5 FM or 5 @M W7. respectively). Ncvtrthelcss, the release was significantly inhibited as compared with the control studies using no inhibitor. as well as with W12. a derivative of W7 with much less inhibitory activity to calmodulin. This result indicates that the involved cnzymc was calmodulin-dependent. Scvanian et al. reported that accumulation of membrane lipid peroxides in the membrane induces phophohpase AZ activation (3). and suggested that factors dccrcasing mcmbranc fluidity may influence phospholipase A: activity (3). Monchilova also showed that decrcascd membrane fluidity leads to augmentation of phospholipase A? activity (7). In B-16 melanoma cells. plasma membrane fluidity decreases immediately after UV-B exposure [ 11, therefore it would be possible for UV-B to influence phospholipase A? activity. All taken together, it is suggested that UV-B irradiation may induce a Ca7+ influx which then stimulates Ca’+-carmodulin-dependent phospholipase AZ to release arachidonic acid and r&ted
156
PROSTAGLANDINS
substances from membrane phospholipids, thereby contributing to the supply of a substrate for inflammatory prostanoids and also contributing to the repair process of UV-B-induced oxidative membrane damage. However, involvement of the phospholipase C-mediated pathway is not completely excluded in this study.
violet irradiation.
3.
Acknowledgements 5,
6.
References 1. Sakanashi, T.. Sugiyama, M., Suematsu, T.. Hidaka, T. and Ogura, R. Delayed alteration of membrane fluidity in intact cultured B-16 melanoma cells affected by ultra-
AND ESSENTIAL FAlTY
ACIDS
Biochem. Int. 12: 314-348, 19X6.
2. Grossman, A. and Wendel. A. Nonreactivity of selenoen-
4.
The authors thank Shiseido Company for the generous gift of B-16 melanoma cells. This works was partly supported by grants from Shiseido company.
LEUKOTRIENES
7.
zyme glutathione peroxidase with enzymatically hydroperoxidized phospholipids. Eur. J. Biochem., 135: 549-552, 1983. Sevanian, A., Muakkassh-kelly. S. F. and Montestruque, S. The influence of phospholipase AZ and glutathione peroxidase on the elimination of membrane lipid pcroxides. Arch Biochem. Biophys., 223: 441-452. 1983. Waite. M.. Scherphof, G. L., Boshouwers, F. M. G. and Van Deenen, L. L. M. Differentiation of phospholipases A in mitochondria and lysosomes of rat liver. J. lipid Res. 10: 411-420, 1969. Franson, R. and Weglicki. W. Phospholipase A activity of lysosomes of rat myocardial tissue. Biochemistry. 11: 472-478, 1972. Deuticke, B.. Heller, K. B. and Haest, C. W. M. Leak formation in human erythrocytes by the radical-forming oxidant t-butyl-hydroperoxide. Biochem. Biophys. Acta.. 858: 169-183, 1986. Monchilova, A.. Petkoua, D. and Kovanov. K. Rat liver microsomal phospholipase A2 and membrane fluidity. Int. .I. Biochem. 18: 659-663, 1986.
Fatty
Acids
(1989) 35. 153-156
Effect of UV-B Irradiation on Release of Arachidonic Acid from B-16 Melanoma cells T. SUEMATSU,
T. HIDAKA,
T. SAKANASHI,
Department of Medical Biochemistry, Kurume, 830, Japan (Reprint request:
M. SUGIYAMA
Kurume University to R. 0.)
School
and Ft. OGURA of Medicine,
67 Asahi-machi,
Abstract - B-16 melanoma cells in culture were prelabeled with (3H)-arachidonate, and exposed to UV radiation. Immediately after irradiation the cells released labeled materials. This UV-stimulated release was inhibited by mepacrine (20 PM) and calmodulin inhibitor W7 (0.5 PM). To determine the influence of extracellular Ca2+ on the UV-stimulated release, experiments were made with media containing various concentrations of Ca”. The release decreased significantly at lower Ca*+ concentrations. These results suggest that Ca*+-calmodulin-dependent phospholipase A2 was involved in UV-stimulated release
of radiolabeled
materials,
possibly
arachidonic
acid and its metabolites,
Introduction
Plasma membranes are among the cell constituents most sensitive to oxidative damage because of their high content of polyunsaturated fatty acids mainly esterified in the 2-position of various cellular phospholipids. In cultured B-16 melanoma cells, lipid peroxides promptly’ increase after UV exposure but 6 hours later the content of lipid peroxides drops to less than the control level (1). This phenomenon suggests that cultured B-16 melanoma cells have some mechanism which reduces lipid peroxides. Phospholipase A2 hydrolyzes ester bounds in the 2-position of membrane phospholipids and releases free fatty acids. There is much evidence that the GSH-peroxidase-GSH-reductase system can not directly act on fatty acyl chains which are esterified in membrane phospholipids (2), and
from the cells.
that phospholipase AZ is involved processes of membrane phospholipids by oxidative stress (3). In this study, B-16 melanoma cells were exposed to UV-B irradiation as tive stress and a release of arachidonic examined. Materials
in repair damaged in culture an oxidaacid was
and Methods
Cell culture
A line of B-16 melanoma cells was a gift from Shiseido Co. Ltd (Tokyo). B-16 melanoma cells were cultured at 37°C in a 5% COd95% air atmosphere in Eagle’s minimal essential medium (Eagle’s MEM, Nissui) supplemented with 10% calf serum (Flow Laboratories). Cells were harvested using 0.02% EDTA / C*+- Mg*+-free
153
1.54
PROSTAGLANDINS
Dulbecco’s phosphate buffered saline ( PBS(-)) and 0.25% tripsin (9/l, v/v). Cells (2 x 105/dish) were plated as monolayers in Q 60 mm dishes, usually 48 hrs prior to experiments. Following experiments were performed when the number of cells reached approximately 1 x 10h/dish. Cell viability was confirmed by the trypanblue exclusion test. Ultraviolet (UV) irradiation of the cells
Approximately 1 x lo6 cells in a monolayer were irradiated with four UV lights (Toshiba, FL-20, SE 30, wave length 290-320 nm UV-B). Release of arachidonic after W-B exposure
acid and its metabolites
After 24 hrs incubation following the cell seeding, the seeding media were removed and the cells were labeled with 0.4 /_Ki [‘Hl-arachidonic acid (“H)AA) (specific activity: 155 Ci/mol, Amersham International) in 4 ml of MEM + 10% calf serum. The labeling media were removed after 24 hr incubation in a 5% CO2/95% air atmosphere, and the cells were washed twice with 1 ml of incubaton media consisting of PBS(-) containing 0.1% glucose, 0.05% fatty acid-free bovine serum albumin and 1 mM CaC&. Then 1.2 ml of the incubation media was added again. After 1 hr incubation ior stabilization, a 0.2 ml portion of the incubation media was removed to measure each initial [‘HI background. Thereafter the dishes were irradiated for 15 min (1600 J/m’). Some of the dishes were protected from the irradiation by being covered with aluminium foil as a control. Immediately after UV exposure, the incubation media were collected from the dishes. The media obtained before and after irradiation were centrifuged at 3000 rpm for 10 min to remove detached cells and mixed with 10 ml of liquid scintillation liquid (toluene containing 35% (V/V) Triton X-100, 5 $I 2,5diphenyloxazole and 0.43 $1 1,4-bis-2,5-phenyloxazole benzene), and then assayed by a liquid scintillation counter (Aloka, LSC-673). Radio activities of [3H]AA and its metabolites released during the 15 min exposure were determined by calculating the difference of radioactivities before and after the exposure. Effects of the following treatments were evaluated by the ratio of the radioactivity from UV-exposed cells to that from UVprotected cells.
LEUKOTRIENES
AND ESSENTIAL FA-I-I’Y ACIDS
Study in few Ca’+ condition
In order to study the effect of extracellular Ca?+ concentration on the release of arachidonic acid and its metabolites stimulated by UV-B, four different media, which contained 1 mM Ca’+, 20 I_LMCa’+, 5 PM (a’+, or 5 PM Ca’+ + 6 PM EGTA respectively, were used. The concentration of Ca’+ was confirmed with a Hitachi atomic absorption spectrophotometer (180-60/80). Inhibitor studies
Effects of phospholipase inhibitor and calmodulin inhibitors on UV-B-induced release of arachidonic acid and its metabolites were examined by the addition of 20 PM mepacrine, 0.5 PM W7 or W12 to the medium, These inhibitors did not affect cell viability after 15 min UV exposure. Results B-16 melanoma cells prelabeled with (“H)AA were exposed to various doses of UV. Radiolabeled material(s) was released into the extracellular medium in a UV-dose dependent fashion. However, cell viability markedly decreased with increasing UV-B dose. The maximum energ which did not affect viability was 16 000 J/m Y, Average release at 16 000 J/m* of UV was significantly higher than that of unexposed controls (p
*= p > 0.05.
1366 * 698 742 f 542
Values are mean + SD.
dpm
*
dpm (n = 13)
Table 2 (I, II, III and IV) shows the influence of lowering the concentration of extracellular Ca’+ on the release of the radiolabeled materials. The UV-stimulated release significantly decreased with decreasing concentration of Cal+. Table 2 (I, V, VI and VII) shows the inhibitory effects of mepacrine (a phospholipase inhibitor) and W7 (a calmodulin inhibitor) on the release
EFFECT OF UV-B IRRADIATION
ON RELEASE
OF ARACHIDONIC
Effects of Extracellular Ca’+ Concentrations, the Calmodulin Inhibitor W7, and Mepacrine on the Total Release of (3H) Arachidonic Acid and Related Materials from B-16 Melanoma Cells
Table 2
Treatment Control fI mM Ca”) 20 /LM (‘a” 5 /.LM Ca” 5 /LM Ca’+ + 6 ELM EGTA Mepacrine20 PM (1 mM Ca”) WI2 0.5 PM (1 mM Ca”) W7 0.5 +M (1 mM Ca”)
UV-induced / protected from UV 2.30 + 1.25 (n = 13) 1.72 1.17 0.94 1.20
rt 2 k k
0.65 0.59 0.59 0.22
(n (n (n (n
= = = =
12) 9)* 6)* 5 6)*
2.43 f 0.81 (n = 7) 1.26 f (1.44 (n = 7)* 1
(UV-treated/protected from UV) indicates the ratio of the radioactivity from UV-exposed cells to that from UVprotected cells. n = number of experiments. * = p < 0.05 as compared with the control. 6 = p < 0.05 as compared with the group treated with 20 /LM Cal+. 1 = p < 0.01 as compared with the group treated with W17.
of the radioactive materials. In the presence of the release was inhibited 20 PM mepacrine, significantly (p
Our confirmations in this study include: (I) UVB irradiation stimulated the release of radiolabeled materials from (3H)AA-prelabeled B-16 melanoma cells; (II) the release of radiolabeled materials was dependent on extracellular Ca’+ concentrations; (III) the release was inhibited by the phospholipase inhibitor, mepacrine, and (IV) the release was inhibited by the specific calmodulin inhibitor, W-7. Phosphoglycerides were the main lipids incorporating radiolabeled AA. The released materials must include not only AA but also eicosanoids and other oxidized lipids derived from AA. The release is most likely due to the activation of phospholipase AZ, which is thought to be an enzyme universally occuring in live cells. Our preliminary experiment also confirmed the activity of this enzyme in B-16 melanoma
ACID FROM B-16 MELANOMA
CELLS
155
homogenate (19.2 5 12.0 nmol oleic acid release / mg protein / hr). Phospholipase Al has been established to be dependent upon intracellular Ca2+ concentrations except in lysosomes in which it is rather inhibited by the ion [4,5]. Our result that the UV-stimulated release was more inhibited at lower extracellular Ca” concentrations suggests that the UV-B irradiation perturbed the plasma membrane in favor of increasing the influx of Ca’+, and resultantly activated the involved enzyme, probably phospholipase A,, in an intracellular-Ca’+-conccntration dependent manner. This view agrees with Deutike. et al, who reported that oxidativc damage by t-butylhydroperoxide formed an ionpermiable leak pathway, probably due to phospholipid peroxidation (6). since UV-B is an oxidative stress already shown to trigger lipid peroxidation in cultured B-16 melanoma cells (1). The fact that mepacrine could markedly inhibit the UV-B activated release strongly suggests that phospholipase A: was involved in the release of radiolabelled materials by UV-B irradiation. As concerns the calmodulin inhibitor. WCwere obliged to use the rather low concentration of W7, because UV-B exposure to cells in the presence of more than 0.5 PM W7 lead to drastically low viability (84% or 36% viability in 0.5 FM or 5 @M W7. respectively). Ncvtrthelcss, the release was significantly inhibited as compared with the control studies using no inhibitor. as well as with W12. a derivative of W7 with much less inhibitory activity to calmodulin. This result indicates that the involved cnzymc was calmodulin-dependent. Scvanian et al. reported that accumulation of membrane lipid peroxides in the membrane induces phophohpase AZ activation (3). and suggested that factors dccrcasing mcmbranc fluidity may influence phospholipase A: activity (3). Monchilova also showed that decrcascd membrane fluidity leads to augmentation of phospholipase A? activity (7). In B-16 melanoma cells. plasma membrane fluidity decreases immediately after UV-B exposure [ 11, therefore it would be possible for UV-B to influence phospholipase A? activity. All taken together, it is suggested that UV-B irradiation may induce a Ca7+ influx which then stimulates Ca’+-carmodulin-dependent phospholipase AZ to release arachidonic acid and r&ted
156
PROSTAGLANDINS
substances from membrane phospholipids, thereby contributing to the supply of a substrate for inflammatory prostanoids and also contributing to the repair process of UV-B-induced oxidative membrane damage. However, involvement of the phospholipase C-mediated pathway is not completely excluded in this study.
violet irradiation.
3.
Acknowledgements 5,
6.
References 1. Sakanashi, T.. Sugiyama, M., Suematsu, T.. Hidaka, T. and Ogura, R. Delayed alteration of membrane fluidity in intact cultured B-16 melanoma cells affected by ultra-
AND ESSENTIAL FAlTY
ACIDS
Biochem. Int. 12: 314-348, 19X6.
2. Grossman, A. and Wendel. A. Nonreactivity of selenoen-
4.
The authors thank Shiseido Company for the generous gift of B-16 melanoma cells. This works was partly supported by grants from Shiseido company.
LEUKOTRIENES
7.
zyme glutathione peroxidase with enzymatically hydroperoxidized phospholipids. Eur. J. Biochem., 135: 549-552, 1983. Sevanian, A., Muakkassh-kelly. S. F. and Montestruque, S. The influence of phospholipase AZ and glutathione peroxidase on the elimination of membrane lipid pcroxides. Arch Biochem. Biophys., 223: 441-452. 1983. Waite. M.. Scherphof, G. L., Boshouwers, F. M. G. and Van Deenen, L. L. M. Differentiation of phospholipases A in mitochondria and lysosomes of rat liver. J. lipid Res. 10: 411-420, 1969. Franson, R. and Weglicki. W. Phospholipase A activity of lysosomes of rat myocardial tissue. Biochemistry. 11: 472-478, 1972. Deuticke, B.. Heller, K. B. and Haest, C. W. M. Leak formation in human erythrocytes by the radical-forming oxidant t-butyl-hydroperoxide. Biochem. Biophys. Acta.. 858: 169-183, 1986. Monchilova, A.. Petkoua, D. and Kovanov. K. Rat liver microsomal phospholipase A2 and membrane fluidity. Int. .I. Biochem. 18: 659-663, 1986.